Alcohol (ethanol) is a major drug of abuse in the U.S. and worldwide, with deaths from alcohol-related cases numbering over a hundred thousand per year. New pharmacotherapies for treating alcohol use disorders are urgently needed. Repeated alcohol consumption alters the neural activity in the brain through ethanol's direct modulation of different types of ion channels. The pharmacological effects of ethanol occur with milliMolar concentrations. In order to understand how ethanol alters brain circuits it is essential to elucidate the molecular and chemical mechanisms underlying ethanol's low affinity modulation of brain proteins. Studies of GIRK channels have provided one of the best examples of modulation by ethanol. GIRK channels are activated directly by ethanol and have been clearly implicated in ethanol-drinking behaviors. Mice lacking GIRK2 subunits self-administer more ethanol than wild-type mice while mice lacking GIRK3 subunits increase binge- drinking of alcohol. These studies suggest that GIRK2 and GIRK3 subunits may differ in their sensitivity to ethanol. GIRK channels possess a pocket for alcohol that has been described at the atomic resolution for GIRK2 channels. However, little is known about the structure of heteromeric GIRK3-containing channels. It is hypothesized that the alcohol pocket in heteromeric GIRK3-containing channels coordinates ethanol better than GIRK2 channels, resulting in a higher sensitivity to alcohol. This grant will determine the chemical and structural differences in alcoho-sensitive properties of GIRK channel subunits using purified homo- and hetero- tetrameric GIRK channels reconstituted into defined lipids with cholesterol, and solving atomic structures of alcohol pockets. The effect of alcohol on microscopic gating properties of purified GIRK channels will also be determined to test the hypothesis that alcohol increases the open channel probability via an increase in the frequency of opening, in contrast to the mechanism of G proteins that stabilize an open state. Lastly, the atomic resolution structure of the GIRK alcohol pocket will be used to identify novel ethanol modulators, using in silico screening. Candidate drugs will be tested using a high-throughput flux assay with purified GIRK channels and then with native GIRK channels expressed in ventral tegmental area dopamine neurons for ex vivo studies. Defining the physical pocket for ethanol is critical for understanding how the low affinit binding of ethanol changes channel activity and affects brain function. Results from this grant could pioneer the development of novel treatments for alcohol abuse and dependence.